The progression of a proliferating eukaryotic cell through the cell-cycle checkpoints is controlled by an array of regulatory proteins that guarantee that mitosis occurs at the appropriate time. Protein phosphorylation is the most common post-translational modification that regulates processes inside the cells, and a large number of cell cycle transitions are regulated by, in addition to protein-protein interactions, the phosphorylation states of various proteins. In particular, the execution of various stages of the cell-cycle is generally believed to be under the control of a large number of mutually antagonistic kinases and phosphatases. A paradigm for these controls is the CDC2 protein kinase, whose activity is required for the triggering of mitosis in eukaryoitc cells (for reviews, see Hunt (1989) Curr. Opin. Cell Biol. 1:268-274; Lewin (1990) Cell 61:743-752; and Nurse (1990) Nature 344:503-508). During mitosis, the CDC2 kinase appears to trigger a cascade of downstream mitotic phenomena such as metaphase alignment of chromosomes, segregation of sister chromatids in anaphase., and cleavage furrow formation. Many target proteins involved in mitotic entry of the proliferating cell are directly phosphorylated by the CDC kinase. For instance, the CDC2 protein kinase acts by phosphorylating a wide variety of mitotic substrates involved in regulating the cytoskeleton of cells, such that entry into mitosis is coordinated with dramatic rearrangment of cytoskeletal elements.
The CDC2 kinase is subject to multiple levels of control. One well-characterized mechanism regulating the activity of CDC2 involves the phosphorylation of tyrosine, threonine, and serine residues; the phosphorylation level of which varies during the cell-cycle (Draetta el al. (1988) Nature 336:738-744; Dunphy et al. (1989) Cell 58:181-191; Morla et al. (1989) Cell 58:193-203; Could et al. (1989) Nature 342:39-45; and Solomon et al. (1990) Cell 63:1013-1024). The phosphorylation of CDC2 on Tyr-15 and Thr-14, two residues located in the putative ATP binding site of the kinase, negatively regulates kinase activity. This inhibitory phosphorylation of CDC2 is mediated at least in part by the wee1 and mik1 tyrosine kinases (Russel et al. (1987) Cell 49:559-567; Lundgren et al. (1991) Cell 64:1111-1122; Featherstone et al. (1991) Nature 349:808-811; and Parker et al. (1992) PNAS 89:2917-2921). These kinases act as mitotic inhibitors, over-expression of which causes cells to arrest in the G2 phase of the cell-cycle. By contrast, loss of function of wee1 causes a modest advancement of mitosis, whereas loss of both wee1 and mik1 function causes grossly premature mitosis, uncoupled from all checkpoints that normally restrain cell division (Lundgren et al. (1991) Cell 64:1111-1122).
As the cell is about to reach the end of G2, dephosphorylation of the CDC2-inactivating Thr-14 and Tyr-15 residues occurs leading to activation of the CDC2 complex as a kinase. A stimulatory phosphatase, known as CDC25, is responsible for Tyr-1 5 and Thr-14 dephosphorylation and serves as a rate-limiting mitotic activator. (Dunphy et al. (1991) Cell 67:189-196; Lee et al. (1992) Mol. Biol. Cell. 3:73-84; Millar et al. (1991) EMBO J 10:4301-4309; and Russell et al. (1986) Cell 45:145-153). Recent evidence indicates that both the CDC25 phosphatase and the CDC2-specific tyrosine kinases are detectably active during interphase, suggesting that there is an ongoing competition between these two activities prior to mitosis (Kumagai et al. (1992) Cell 70:139-151; Smythe et al. (1992) Cell 68:787-797; and Solomon et al. (1990) Cell 63:1013-1024). This situation implies that the initial decision to enter mitosis involves a modulation of the equilibrium of the phosphorylation state of CDC2 which is likely controlled by variation of the rate of tyrosine dephosphorylation of CDC2 and/or a decrease in the rate of its tyrosine phosphorylation. A variety of genetic and biochemical data appear to favor a decrease in CDC2-specific tyrosine kinase activity near the initiation of mitosis which can serve as a triggering step to tip the balance in favor of CDC2 dephosphorylation (Smythe et al. (1992) Cell 68:787-797, Matsumoto et al. (1991) Cell 66:347-360; Kumagai et al. (1992) Cell 70:139-151; Rowley et al. (1992) Nature 356:353-355; and Enoch et al. (1992) Genes Dev. 6:2035-2046). Moreover, recent data suggests that the activated CDC2 kinase is responsible for phosphorylating and activating CDC25. This event would provide a self-amplifying loop and trigger a rapid increase in the activity of the CDC25 protein, ensuring that the tyrosine dephosphorylation of CDC2 proceeds rapidly to completion (Hoffmann et al. (1993) EMBO J 12:53).
Studies of the meiotic cell cycle have likewise demonstrated the role of inhibitory phosphorylation of Tyr-15 and/or Thr-14 on induction of meiosis, and indicate that activation of meiotic cyclin dependent kinases (CDK)/cyclin complexes (known as maturation promoting factor, MPF) is mediated by the antagonistic actions of the Wee1 protein kinase and the CDC25 tyrosine phosphatase. MPF, like the mitotic CDK/cyclin complexes, is activated by removing the inhibitory tyrosine phosphate from a pool of preMPF. In accord with this scheme, preMPF complexes can be isolated from unstimulated oocytes and converted into active MPF in vitro by treating them with purified CDC25. For instance, in Xenopus oocytes that are blocked in prophase of the first meiotic division, CDC2 kinase is complexed with mitotic cyclins but is present in an inactive, Thr14 and Tyr15 phosphorylated state. Upon stimulation with progesterone, or insulin, oocytes undergo the transition into meiosis (maturation), that is, effect dephosphorylation of CDC2 on the Thr14 and Tyr15 residues and thus the activation of the CDC2 histone H1 kinase (Dunphy et al. (1989) Cell 58:181-191; and Gautier et al. (1989) Nature 339:626-629). As above, the initiation of maturation probably reflects a change in the balance between the opposing activities of CDC25 and Wee 1.
In addition to internal regulatory signals, such as those networks which guarantee that the successive events of each cell-cycle occur in a faithful and punctual manner, cell proliferation may also be regulated by chains of events that begin with the interaction of growth factors with specific growth factor receptors located in the plasma membrane of the cell. The binding of a growth factor by its cognate receptor induces the receptor to generate signals inside the cell, and, by propagation along signal transduction pathways, can result in the activation of a variety of intracellular enzymes, mainly protein kinases and phosphatases. Changes in protein phosphorylation, mediated by the signal transduction process, can lead to the transcription of early response genes that encode transcription factors, which in turn induce the transcription of delayed response genes. The products of the delayed response genes include cell cycle regulatory proteins, such as G.sub.1 cyclins and cyclin dependent kinases. However, certain aspects of growth factor induction have not previously been elucidated. For instance, it is not apparent from the literature how it is that growth factors and the signals they induce antagonize the ability of tumor suppressor gene products, like the retinoblastoma protein (Rb), to inhibit the transcription of early response genes.